Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia.
Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.
Hornung
Ellen
E
Georg-August-University Göttingen, Albrecht-von-Haller Institute for Plant Sciences, Department for Plant Biochemistry, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
Kunze
Susan
S
Liavonchanka
Alena
A
Zimmermann
Grit
G
Kühn
Diana
D
Fritsche
Kathrin
K
Renz
Andreas
A
Kühn
Hartmut
H
Feussner
Ivo
I
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005973:Glutamine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006880:Hydroxy Acids / Q000737:chemistry; D008084:Lipoxygenase / Q000737:chemistry*; D008969:Molecular Sequence Data; D029777:Momordica charantia / Q000196:embryology / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D012639:Seeds / Q000201:enzymology*; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000502:physiology
D006880:Hydroxy Acids; D005973:Glutamine; D008084:Lipoxygenase